1. Technical Field
The present invention relates to the cleavage of mRNA directing the expression of the enzyme steroid 5.alpha.-reductase in mammalian cells, including human cells. More specifically, the present invention provides ribozymes capable of selectively cleaving such mRNA, thereby advantageously reducing the expression of 5.alpha.-reductase by cells exposed to such a ribozyme. The invention also provides methods and compositions for administering such ribozymes to cells, including methods and compositions for topically administering a 5.alpha.-reductase-mRNA-specific ribozyme to, inter alia, hair follicle cells for the treatment of androgenic alopecia.
2. Description of the Background Art
The microsomal enzyme steroid 5.alpha.-reductase (EC1.3.99.5) catalyzes the conversion of 4-ene-3-keto-steroids to the corresponding 5.alpha.-dihydro-3-keto steroids in human and other mammalian tissues. The conversion of testosterone to the more-potent androgen dihydrotestosterone ("DHT") is one of the best-characterized and best-known roles of this enzyme. DHT is considered to be the most potent androgen and to be responsible for differentiation of the male external genitalia and prostate as well as for virilization at puberty. See Labrie et al., Endocrinology 131:3, 1571-1573 (1992).
Of the several organs that produce androgens, the testes produce these hormones in the greatest amounts. Centers in the brain exert primary control over the level of androgen production and, as described in greater detail below, numerous physical manifestations and disease states result when ineffective production control results in excessive androgen hormone production.
The enzyme 5.dbd.-reductase plays a major role in a variety of androgen-specific diseases, dysfunctions and physical conditions including but not limited to prostate cancer, benign prostatic hyperplasia, acne, seborrhea, hirsutism and androgenic alopecia including male pattern baldness. A variety of chemical inhibitors of this enzyme have been proposed for various therapeutic uses. See for example McConnell et al., J. Clin. Endocrinol. Metab. 74:505-508, which describes the use of finasteride for suppressing prostatic DHT in men with benign prostatic hyperplasia. The development of inhibitors of 5.alpha.-reductase has been hampered, however, by the absence of sufficient knowledge of the structure of the protein and by the very low levels of expression even in androgen-responsive tissues. See Andersson, S. et al., PNAS USA 87:3640-3644 (May, 1990). Moreover, in the case of finasteride, development of impotence is an undesirable side effect of this agent. Thus, there remains a need for improved effective and specific means for suppressing the production of DHT in patients in need thereof.
Benign prostatic hypertrophy (BPH) is a common problem in elderly males due to increased conversion of testosterone to DHT via increased activity of 5-.alpha. reductase, with significant clinical morbidity including obstruction, urinary retention, and difficulty in initiating or terminating urinary stream. Management of BPH is predominantly surgical with the risk of inherent complications. Recently, finasteride, a specific inhibitor of 5.alpha.-reductase, has shown some promise in the treatment of BPH despite the development of impotence in some patients (McConnell et al. as above). Therefore, development of an improved effective and specific means for suppressing the production of DHT via the 5-reductase pathway will find utility beyond the treatment of androgenic alopecia.
It is well established that, in androgenic alopecia (including male pattern baldness), an accumulation of androgens, especially DHT, in hair follicle cells plays a significant role. See Hamilton, J. B., Am. J. Anat., 71:451-480 (1942); Mooradian et al., Endocr. Rev., 8:1-28 (1987); Cunha et al., Endocr. Rev., 8:338-362 (1987). Testosterone circulating in the bloodstream serves as a prohormone for the more-active DHT in certain tissues. Testosterone is converted to the more-potent androgen DHT in the epidermis (Harris et al., Proc. Natl. Acad. Sci. USA 89:10787-10791 (1992)), this conversion being catalyzed by the 5.alpha.-reductase enzyme. Male pattern baldness is characterized by the shrinkage of hair follicles on the crown of the head, ultimately leading to such follicles becoming dormant. Such shrinkage of hair follicles is largely due to the action of testosterone and DHT on the hair follicle cells. Individuals showing male pattern baldness begin to lose their hair early in life--often in their twenties--and may employ a variety of techniques with hopes of reversing the condition, often at great expense but with minimal effect.
It had been postulated that the presence of accumulations of DHT in the skin gives rise to the symptoms of both acne and androgenic alopecia including male pattern baldness. The presence of increased levels of 5.alpha.-reductase in hair follicles of patients, both male and female, exhibiting androgenic alopecia has been demonstrated. M. E. Sawaya reported (Dermatology, Progress & Perspectives, Proceedings of the 18th World Congress of Dermatology, pp. 202-203 (1992), Parthenon Pub. Group) that frontal follicles of women exhibiting androgenic alopecia contained almost twice the 5.alpha.-reductase level of occipital follicles, and that frontal follicles of men exhibiting androgenic alopecia contained more than twice the 5.alpha.-reductase level of occipital follicles. Thus, there is today little question that the conversion of testosterone to DHT, catalyzed by the enzyme 5.alpha.-reductase, plays an important role in androgenic alopecia.
With respect to acne, sebum secretion is on average higher in patients with acne vulgaris than their normal counterparts. Therefore, compounds which reduce sebum production have therapeutic potential. Among such agents are anti-androgens, including 5.alpha.-reductase inhibitors (Cuncliffe and Schuster, Lancet, 1:685-687 (1969)).
Among the several functions of RNA within cells is its role of enzymatic catalysis. Ribozymes are catalytic RNA's that have the capacity to specifically base-pair with, cleave and thus functionally destroy a an RNA sequence. See, generally, Cech and Bass, Ann. Rev. Biochem. 55:599-629 (1986). In nature, these RNA catalysts function in cis, cleaving a portion of the same RNA strand upon which they reside. The isolation of the catalytic portions of such RNA's into discreet molecules, or "trans-acting" ribozymes, however, has provided useful reagents having riboendonuclease activity. See Haseloff & Gerlach, Nature 334:585-591 (1988).
The class of "hammerhead" ribozymes, for example, comprises such trans-acting reagents. Hammerhead ribozymes cleave an exogenous RNA template immediately 3' to a GUX sequence (wherein X is C, A or U). Hammerhead ribozymes are comprised of a conserved catalytic core flanked by sequences complementary to the desired target RNA sequence to confer target specificity (Haseloff et al., supra.). Even more recently, the ability to engineer ribozymes in a variety of ways, for improved cleavage specificity and enhanced catalytic turnover, for example, has been documented. Thus, catalytic RNA's (which may also contain one or more deoxyribonucleotide bases), generically referred to herein as "ribozymes," which are capable of site-specific cleavage of exogenous RNA (and sometimes DNA) strands present an important mechanism for the modulation of gene expression in mammalian cells.
Two human 5.alpha.-reductase enzymes have been identified, and their genes have been isolated and sequenced. The cDNA and amino acid sequences of human type I 5.alpha.-reductase appears in Andersson, S. et al., supra, which publication indicated that the amino acid sequence has been deposited in the GenBank data base under accession no. M32313. Andersson et al. reported the discovery of a second human (type II) 5.alpha.-reductase in Nature, 354:159-161 (1991), which, unlike the type I enzyme, is the major 5.alpha.-reductase found in prostatic tissues and is sensitive to finasteride. The isolation and structural characterization of the human type II 5.alpha.-reductase gene, including its sequence, were reported by Labrie et al. in Endocrinology 131:3, pp. 1571-1573 (1992).
Accordingly, it is an object of the present invention to provide a means for reducing the production or accumulation of DHT in mammalian tissues, including human tissues.
Another object of the present invention is to reduce the production or accumulation of DHT in tissues, especially epidermal tissue, by reducing the expression of the microsomal steroid enzyme 5.alpha.-reductase in epidermal tissue.
An additional object of the invention is to provide a pharmaceutical composition for topical application which contains a ribozyme capable of cleaving mRNA coding for 5.alpha.-reductase.
Yet another object of the invention is to provide a method for treating androgenic alopecia by the topical application of a ribozyme capable of cleaving mRNA coding for 5.alpha.-reductase.
A still further object of the invention is to provide reagents useful for the in vitro study of the biosynthesis of mammalian 5.alpha.-reductase enzymes, including the human type I and type II enzymes. Such reagents contain amounts of a 5.alpha.-reductase-mRNA-specific ribozyme effective to reduce or halt entirely the production of 5.alpha.-reductase by cells exposed to them.